In many medical and biological fields, cell counting is a critical aspect of laboratory analysis. Cells can be counted manually, for example by simple counts on slides or in a counting chamber, or automatically using, for example, flow cytometry or image analysis. It is this last area, image analysis, that has gained in usage in recent years, given the flexibility in image analysis systems and the relative low cost as compared to typical flow cytometers.
Image analysis systems utilize both transmitted light and fluorescence to produce static images of viable and/or dead cells in a sample. Optics usually comprise an objective lens in optical communication with a detection system, typically a charge-coupled device (CCD) camera or a complementary metal-oxide-semiconductor (CMOS) camera. Images produced by the camera are then transmitted to a computer for analysis by software designed to count cells in the bright-field or fluorescence images or alone or in combination.
U.S. Pat. Nos. 8,883,491 and 9,046,489 disclose cell counters that utilize the combination of bright-field light and fluorescent light for cell counting. In U.S. Pat. No. 8,883,491, a cell counting system takes images of a static population of cells in a sample that has been loaded into a chamber having a fixed height. Because the system utilizes a covered chamber having a fixed height, cell concentration can be determined from the cell count. U.S. Pat. No. 9,046,489 discloses a fluorescence imaging device that comprises a fluorescence light source, a light source configured to emit white light or monochromatic light, an excitation filter, a dichroic mirror configured to transmit excitation light transmitted by the excitation filter and reflect light fluorescence emission light, an objective lens, an emission filter configured to transmit fluorescence emission light of a predetermined wavelength, and a detector.
A significant disadvantage of automated image analysis systems described above is their use of cartridges for sample analysis. Similar to the so-called “razor blade model” (except without the initial low cost purchase of the base item), these cartridges are single use, disposable items that lead to high costs for the user. Further, these cartridges tend to be designed for specific devices, and cannot be used with cell counters from other manufacturers or even other devices from the same manufacturer.
A fixed path length over a defined surface area establishes a fixed sample volume for analysis. The cell density of the sample may be too low or too high for accurate cell counting thereby limiting the effective detection range of the apparatus. Additionally cells can clump on top of each other in a fixed path length chamber excluding the cells that are out of the field of focus from being counted.
Reducing the path length to less than twice the diameter of the cell will force all cells in a sample into the same focal plane.
Thus, there remains a need for cell counters and methods of use thereof that reduce cost, provide greater analysis flexibility, and improve data quality.